【作者】 王景玲；

【导师】 侯金林； 陈金军；

【作者基本信息】 南方医科大学 ， 内科学， 2010， 博士

【摘要】 研究背景：乙型肝炎病毒(Hepatitis B virus, HBV)感染呈世界性流行,我国属HBV感染的地方性高流行区。与欧美国家不同的是,我国的大多数慢性乙型肝炎患者是在围产期或婴幼儿期感染,因感染时免疫系统发育尚不完善,分别有90%和25%发展成慢性感染,其HBV感染的自然史一般可分为三个期：免疫耐受期、免疫清除期和低复制期,部分专家将在低复制期发生的肝炎再活动定义为第四期,即再活动期。在免疫清除期,随着血清丙氨酸氨基转移酶(alanine aminotransferase, ALT)水平的波动,血清HBeAg合成下降,抗-HBe产生增加,此期的炎症活动有可能会导致自发的HBeAg血清学转换(HBeAg消失及抗-HBe出现)。事实上,在HBV慢性感染的过程中,机体产生的抗-HBe抗体可以和HBeAg在很长的一段时间里(数月至数年)以免疫复合物的形式存在。目前,根据HBeAg状态可将慢性乙型肝炎分为两种类型：HBeAg阳性的慢性乙型肝炎和HBeAg阴性的慢性乙型肝炎。HBeAg阳性的慢性乙型肝炎往往伴随着高水平的病毒复制,而HBeAg阴性的慢性乙型肝炎通常表现为进展性的肝损害,血清ALT以及HBV DNA低水平波动。HBV C基因分为前C区和C区,编码的核心蛋白有两种形式：核壳蛋白(HBcAg,为病毒核壳的结构成分)和分泌性的HBeAg。血清HBeAg滴度受很多因素影响,其中前C/C区变异为其中的一个重要因素。前C/C区变异在转录水平和翻译水平双重层面上影响HBeAg的分泌。在转录水平,基本核心启动子区(basic core promoter, BCP) T1762/A1764变异可使mRNA转录水平下降至野生株的1/5至1/3；在翻译水平,前C区A1896变异使得前C第28位密码子由TGG突变为TAG(终止密码子),导致HBeAg前体翻译提前终止。HBeAg阴性慢性乙型肝炎患者中T1762/A1764及A1896变异比例较高,而HBeAg阳性慢性乙型肝炎患者血清样本中也可发现T1762/A1764及A1896变异,其变异株/野生株比例与血清HBeAg水平相关。慢性HBV感染的血清标志物包括HBsAg、抗-HBs、HBeAg、抗-HBe及抗-HBc。五种血清标志物不同的组合模式代表着不同的疾病状态,在慢性乙型肝炎的临床诊断与治疗评价中发挥重要作用。临床中,常见的血清标志物组合模式为HBsAg、HBeAg及抗-HBc同时阳性或者HBsAg、抗-HBe及抗-HBc同时阳性。除此之外,还存在着一些少见模式,如HBsAg与抗-HBs同时阳性、HBeAg及抗-HBe同时阳性等。HBeAg/抗-HBe同时阳性的慢性乙型肝炎在临床免疫清除期患者中经常看到,然而到目前为止,尚无针对此类HBV感染人群的系统性研究。研究目的：本研究目的在于调查HBeAg/抗-HBe双阳性慢性乙型肝炎患者的流行率；分析其人口学、临床指标、病理学、血清学及病毒学特征；并探讨血清HBeAg和抗-HBe同时阳性的可能机制。研究方法：1、收集2002年1月至2009年7月在南方医院感染内科住院的慢性乙型肝炎患者的人口学信息、临床和实验室资料。纳入标准为：1、住院期间诊断为慢性HBV感染,包括慢性乙型肝炎、乙型肝炎肝硬化代偿期(Child-PughA级)或乙型肝炎肝硬化失代偿期(Child-Pugh B或C级)；2、年龄≥14岁；3、入院前未进行过抗HBV治疗；4、入院前12个月内血清ALT水平间断或持续异常。排除标准为：合并其他病毒感染(如：甲、丙、丁、戊型肝炎病毒或人类免疫缺陷病毒等)；其他原因导致的肝炎(自身免疫性肝病、酒精性肝病、药物性肝损害等)；原发性或继发性肝癌以及由于使用免疫抑制剂或放射治疗等导致的HBV再激活患者。2、根据HBeAg/抗-HBe状态(阴性或阳性)将纳入患者分组,比较各组间人口学、临床指标、病理学、血清学特征。3、根据]HBeAg或抗-HBe检测值(S/CO,雅培i2000或AXSYM)分别将HBeAg单阳性或抗-HBe单阳性慢性乙型肝炎患者各自分为低、中、高滴度三亚组,首先横向比较分析不同HBeAg滴度或抗-HBe滴度单阳性患者亚组间的临床特点。若低、中、高三亚组间临床特点存在差异,则将低滴度单阳性患者与HBeAg/抗-HBe双阳性患者进行纵向比较,分析两组间临床特点的差别。4、对于纳入的HBeAg/抗-HBe双阳性慢性乙型肝炎患者,从本室保存的血清库中查找并调出患者住院时冻存的血清,并通过抽提血清HBV DNA、巢式PCR扩增及直接测序检测HBV的基因型以及基本核心启动子区、前C区和C区变异情况。5、进行HBeAg/抗-HBe双阳性模式的体外模拟实验。第一步,将3对临床血清(HBeAg单阳性血清及抗-HBe单阳性血清各一份为一对)按1：1体积比混合,37℃孵育(以下同)3小时后使用雅培i2000检测HBeAg/抗-HBe。如检测结果为抗-HBe阳性则减少下一次混合时抗-HBe阳性血清所加入的比例,再次混合、孵育、检测,依次循环,直到能够检测到HBeAg及抗-HBe同时阳性。此时加入的HBeAg与抗-HBe单阳性血清的比例,即“合适比例”。根据“合适比例”适当调整多个混合比例,混合后孵育,检测HBeAg及抗-HBe滴度。使混合血清同时具有较高HBeAg及抗-HBe滴度的“合适比例”确定为“最适比例”。第二步,以每对血清的最适比例混合两血清,然后使用小牛血清将混合血清稀释至10～500倍,孵育3小时后进行HBeAg/抗-HBe检测。第三步,以每对血清的最适比例混合两血清,将孵育时间延长至12及24小时,然后进行HBeAg/抗-HBe检测。研究结果：1、纳入的1624例慢性乙型肝炎患者中,169例(10.4%)为HBeAg/抗-HBe双阳性患者,887例(54.6%)为HBeAg单阳性患者,556例(34.2%)为抗-HBe单阳性患者,12例(0.7%)为HBeAg/抗-HBe双阴性患者。因HBeAg/抗-HBe双阴性患者所占比例较小(<1%),未进行进一步分析。2、HBeAg单阳性组(n=887例)、HBeAg/抗-HBe双阳性组(n=169例)及抗-HBe单阳性组(n=556例)三组间的年龄水平及血清HBV DNA水平差异显著(年龄：F=90.180,P<0.001；HBV DNA:F=217.282,P<0.001).HBeAg/抗-HBe双阳性患者的年龄(35.2±11.0岁)及血清HBV DNA水平(5.8±1.5 logcopies/mL)均介于HBeAg单阳性患者(32.0±11.3岁；6.6±1.2 log copies/mL)与抗-HBe单阳性患者(41.0±13.1岁；5.2±1.2 log copies/mL)之间；三组间两两比较,差别均具有统计学意义。三组中双阳性患者的血清ALT水平最高(χ2=12.656,P=0.002),血清总胆红素水平、血清TBIL≥34.2μmol/L的患者比例、肝脏病理炎症分级及纤维化分期均较HBeAg单阳性组高,而与抗-HBe单阳性组相当。3、HBeAg/抗-HBe双阳性组HBeAg滴度显著低于HBeAg单阳性组(Z=-33.934,P<0.001),HBeAg/抗-HBe双阳性组抗-HBe滴度也显著低于抗-HBe单阳性组(Z=-32.039,P<0.001)。双阳性患者的HBeAg滴度及抗-HBe滴度均较低,HBeAg和抗-HBe的中位数(四分位数间距范围)分别为4.2(1.8～9.6)S/CO和0.54(0.27～0.72)S/CO,绝大多数接近于各自的检测临界值。4、根据HBeAg检测值将HBeAg单阳性慢性乙型肝炎患者分为低(1.0<HBeAg≤130.9 S/CO).中(130.9<HBeAg≤293.7 S/CO)、高(HBBeAg>293.7 S/CO)HBeAg滴度三亚组。三亚组间年龄差别显著(F=18.604,P<0.001),以低HBeAg滴度亚组年龄最大(34.8±11.6岁),而高HBeAg滴度亚组年龄最小(29.3±10.1岁)；三亚组间血清HBV DNA水平差别显著(F=64.725,P<0.001),以低HBeAg滴度亚组最低(6.0±1.2 log copies/mL),高HBeAg滴度亚组最高(7.0±1.0 log copies/mL)；三亚组间血清ALT水平差异显著(χ2=9.962,P=0.007),低滴度亚组血清ALT水平在三亚组中最低【中位数(四分位数间距范围),118(47-308)U/L】,中滴度亚组最高【165(75-338)U/L】。另外,低滴度亚组的血清总胆红素水平、肝脏病理炎症分级及纤维化分期在三亚组中最高。5、将低HBeAg滴度亚组与HBeAg/抗-HBe双阳性组比较分析后发现,仅HBeAg/抗-HBe双阳性组血清ALT水平高于低HBeAg滴度组(Z=-2.663,P=0.008),人口学指标及其他临床指标间无差别。6、根据抗-HBe检测值将抗-HBe单阳性乙型肝炎患者分为低(0.105<抗-HBe<1.000 S/CO)、中(0.064<抗-HBe≤0.105 S/CO)、高(抗-HBe≤0.064 S/CO)抗-HBe滴度三亚组后发现,三亚组间血清总胆红素水平有差别(χ2=8.037,P=0.018),以低滴度抗-HBe亚组最高；不同滴度抗-HBe亚组间的平均年龄、血清ALT水平、血清HBV DNA水平及肝脏病理炎症分级及纤维化分期的无差别。7、成功复温的135例HBeAg/抗-HBe双阳性血清中,有134例巢式PCR扩增阳性并成功测序。其中95例(70.9%)为HBV基因B型感染,其余均为HBV基因C型。110例(82.1%)双阳性患者感染了BCP区T1762/A1764和/或前C区A1896终止密码子变异株。8、在HBeAg/抗-HBe同时阳性模式的体外模拟实验中,混合血清37℃孵育3小时后,进行HBeAg及抗-HBe检测(雅培i2000),在三对中均能找到HBeAg及抗-HBe同时阳性的“最适比例”。混合血清经小牛血清稀释至一定比例,HBeAg及抗-HBe仍可同时检测到；如稀释倍数超出一定范围,HBeAg及抗-HBe均检测不到。另外,将孵育时间延长至24小时,HBeAg及抗-HBe仍为同时阳性。与临床双阳性患者血清学特点相似,混合血清HBeAg及抗-HBe滴度也较低,接近于检测的临界值。结论：1、在未行抗病毒治疗的住院慢性乙型肝炎患者中,HBeAg/抗-HBe同时阳性患者并不罕见(占10.4%)。2、HBeAg/抗-HBe双阳性慢性乙型肝炎为慢性HBV感染的自然史中的一个特殊阶段,与更加严重的肝脏疾病相关,此期肝脏病变累积较重。3、机体产生的HBeAg和抗-HBe之间的合适比例为检测出HBeAg和抗-HBe同时阳性的原因之一。4、将HBeAg和抗-HBe同时纳入分类标准可细化分类CHB患者；在HBeAg单阳性患者中,不同HBeAg滴度亚组具有不同的临床特征,HBeAg/抗-HBe双阳性患者与低滴度的HBeAg单阳性患者临床特征相似。更多还原

【Abstract】 BACKGROUND:Chronic infection with hepatitis B virus (HBV) is a significant health problem worldwide and a major cause of morbidity and mortality. China is a high-endemic area, where the majority of hepatitis B virus infections are acquired perinatally or during early childhood. The natural history of perinatal HBV infection is generally divided into three phases according to liver damage and the status of hepatitis B e antigen (HBeAg) and serum HBV-DNA loads:immune tolerant phase, immune clearance phase, and inactive residual phase; hepatitis B re-activation can occur in some patients with inactive disease, which is defined as the fourth phase (re-activation phase). After progression to chronic HBV infection, HBeAg is initially positive in most cases during the immune tolerance phase. Transition to the immune active phase with activation of disease can be abrupt, usually followed by the loss of HBeAg and the development of antibody to hepatitis B e antigen (anti-HBe) (HBeAg seroconversion), sooner or later. In fact, anti-HBe antibody was produced for years and was present in the form of HBeAg-containing immune complexes.The HBeAg status distinguishes two categories of chronic HBV infection. HBeAg positive CHB is accompanied by high-level HBV replication; however, patients with HBeAg-negative chronic HBV infection tend to have progressive liver injury, fluctuating alanine aminotransferase (ALT) activity, and lower levels of HBV DNA.The precore/core gene contains two regions, the precore and core, each containing an initiation (AUG) codon. This gene encodes the core nucleocapsid protein (HBcAg, which is important in viral packaging) and hepatitis B e antigen (HBeAg). The synthesis of HBeAg is doubly controlled at transcription and translation levels. At the transcriptional level, mutations in the basal core promoter (BCP) at nt1762 and nt1764 (resulting in diminished production of HBeAg to 1/5～1/3) were well defined. At the translational level, the mutation at nt1896 caused a stop codon in the precore Open Reading Frame and resulted in the cession of HBeAg expression. High rate of mutations in nt1762/1764 and/or nt1896 were found in HBeAg negative CHB patients, however, these mutations could also be found in HBeAg positive CHB patients and were correlated with serum HBeAg titers.The HBV serum markers include the surface antigen (HBsAg), the e antigen, the surface antibody (anti-HBs), the e antibody and the core antibody. The mode of these markers’ combination is associated with different clinical status, which is helpful for the diagnosis of CHB and antiviral treatment evaluation. HBsAg, HBeAg and anti-HBc positive mode or HBsAg, anti-HBe and anti-HBc positive mode is common in clinical practice. However, a special serological pattern of chronic hepatitis B with concurrent HBeAg and anti-HBe was sometimes seen in clinical practices. A few concurrent patients had been observed in several studies previously, but not been studied in detail to date. Little is known about the clinical and virological features and the mechanisms elucidating this unique serological pattern of HBV e system.AIMS:The aims of this study were to determine the prevalence, analyze the clinical and virological characteristics of patients with and explore the mechanism for the concurrent detection of HBeAg and anti-HBe. METHODS:1, Ranged from January 2002 to July 2009, a Chinese cohort of consecutive inpatients from the Hepatology Unit, Nanfang Hospital was studied. All patients fulfilled the following criteria:1) Diagnosed with chronic HBV infection related liver disease, compensated (Child-Pugh class A) or decompensated cirrhosis (Child-Pugh class B or C); 2) Aged not less than 14 years old; 3) Antiviral treatment-naive prior to admission, and 4) Persistent or intermittent elevation of alanine aminotransferase (ALT) serum levels within 12 months before entry. The exclusion criteria were: coinfection with other hepatitis viruses, human immunodeficiency virus or other causes of hepatitis (autoimmune hepatitis, alcoholic liver disease, etc.). Patients with hepatocellular carcinoma or HBV reactivation by chemotherapy or identified immune suppression therapy were also excluded.2, The patients included were grouped according to serum HBeAg and anti-HBe status. The difference of demography, clinical features, pathology, serology and virology among groups were analyzed.3, The HBeAg or anti-HBe mono-positive patients were stratified into three subgroups according to the HBeAg or anti-HBe titers and features of the three subgroups were analyzed, respectively.4, For the recovered 135 sera samples from concurrent patients, viral genotype, basic core promoter and precore mutations were determined by direct sequencing.5, To explore the possible mechanisms for concurrence of HBeAg and anti-HBe, in vitro simulation of concurrent HBeAg/anti-HBe was performed with three pairs of HBeAg-positive and anti-HBe-positive serum samples, which were randomly selected and paired off. Before analyzing using the Abbott i2000 instrument, sera were mixed at variable ratios between paired samples, vortexed for 10 seconds and incubated at 37℃for 3 hours. The first parameter examined was the optimal ratio between each pair of sera, at which HBeAg and anti-HBe could be concurrently observed. Briefly, sera in each pair were mixed with equal volume to determine whether antigen or antibody was in excess. Then a second mixture was made by decreasing the volume of the serum with the excess marker. More mixed samples were prepared according to the results already obtained until HBeAg and anti-HBe were concurrently observed. The second issue was to test whether concurrence was correlated with the initial concentration of HBeAg and anti-HBe. Mixtures with the optimal ratio were prepared without dilution or with a dilution factor from 10 to 500 before incubation. The third issue was to detrmine the effect of extending the incubation time on concurrent detection. The mixtures with the optimal ratio for each pair were incubated for12 or 24 hours at 37℃.RESULTS:1, A total of 1624 chronic hepatitis B patients fulfilled the study criteria, of which 12 HBeAg/anti-HBe dual negative patients were not further analyzed. The prevalence of patients with concurrent detection of both markers was 10.4%.2, For the age and HBV DNA level, there were significant difference among the three groups (age:F=90.180, P<0.001; HBV DNA:F=217.282, P<0.001). The mean age of the concurrent patients (n=169,35.2±11.0 y) fell in between the HBeAg-positive (n=887,32.0±11.3 y) and anti-HBe-positive (n=556,41.0±13.0 y) groups. The mean HBV-DNA levels (5.8±1.5 log10 copies/mL) of the concurrent group also fell in between those in the HBeAg-positive (6.6±1.2 log) and anti-HBe-positive (5.2±1.2 log) groups. Concurrent patients were associated with the highest ALT levels [166 (65-460) IU/L] compared with HBeAg-positive [144 (65-314) IU/L] and anti-HBe-positive [104 (51-299) IU/L] group (χ2= 12.656, P= 0.002). Percentages of patients with total bilirubin levels over 34.2μmol/L, which is taken as a sign of liver dysfunction, were significantly higher in the concurrent group (47.4%) and anti-HBe-positive group (46.5%) than that in HBeAg-positive patients (25.5%). Thirty five (20.7%) concurrent patients were diagnosed as decompensated cirrhosis, which were comparable to anti-HBe-positive group and were higher than that of HBeAg-positive group. Liver histology evaluations showed that inflammation grades and fibrosis stages were higher in concurrent than in HBeAg-positive patients, but both were comparable to anti-HBe-positive group.3, The median HBeAg titer in the concurrent group was lower than that of the HBeAg-positive group (Z=-33.934, P<0.001). Similarly, the anti-HBe concentration was lower than that of the anti-HBe-positive group (Z=-32.039, P< 0.001). HBeAg and anti-HBe titers (median and interquartile range) in concurrent group were 4.2 (1.8-9.6) S/CO and 0.54 (0.27-0.72) S/CO, most of the HBeAg and anti-HBe titers in the concurrent group were much close to their respective cutoff values than those of the HBeAg-positive or anti-HBe-positive alone patients, respectively.4, The HBeAg mono-positive patients were stratified into three subgroups according to the HBeAg titers (The low titer subgroup,1.0 S/CO< HBeAg≤130.9 S/CO; The medium titer subgroup,130.9< HBeAg≤293.7 S/CO; the high titer subgroup, HBeAg> 293.7 S/CO). The low titer subgroup were associated with the oldest age (F=18.604, P< 0.001), the lowest serum HBV DNA level (F=64.725, P<0.001) and the most profound liver damage. The serum ALT level was the lowest among the three subgroups(χ2=9.962, P=0.007).5, The serum ALT level of concurrent patients was higher than the low HBeAg titer subgroup (Z=-2.663, P=0.008). However, the other features (such as age, HBV DNA, etal.) were comparable between the two groups.6, The anti-HBe mono-positive patients were also stratified into three subgroups according to the anti-HBe titers (The low titer subgroup,0.105 S/CO< anti-HBe< 1.00 S/CO; The medium titer subgroup,0.064 S/CO< anti-HBe≤0.105 S/CO; the high titer subgroup, anti-HBe≤0.064 S/CO). The serum TBil level was highest in low anti-HBe titer subgroup and was lowest in high anti-HBe titer subgroup (χ2= 8.037, P=0.018). However, there was no other difference of demography, the clinical features, the pathology among the three anti-HBe subgroups.7, The HBV core promoter and precore region was successfully amplified and sequenced in 134 cases. For the cases successfully sequenced,110/134 (82.1%) harbored T1762/A1764 or/and A1896 mutants. Of the 134 individuals,95 (70.9%) carried genotype B and 39 (29.1%)C.8, The in vitro simulation showed that HBeAg and anti-HBe could be concurrently observed provided an optimal ratio (HBeAg to anti-HBe) was chosen. Once an optimal ratio was chosen, diluted (dilution factors were 10-500) samples were prepared and concurrent detection was observed at a certain concentration of HBeAg and anti-HBe, as well as dual negative status with excessive dilution. When incubated at 37℃for 12 or 24 hours, HBeAg and anti-HBe could still be concurrently observed.CONCLUSIONS:In antiviral treatment-naive patients, concurrence of HBeAg and anti-HBe was not uncommon, and such patients had profound liver disease. HBeAg mono-positive chronic hepatitis B patients were of heterogeneous patients with different HBeAg titer, there were clinical difference among the three subgroups. The HBeAg/anti-HBe double positive group and the low titer HBeAg-positive subgroup may be in the similar stage during the infection history. An optimal ratio between HBeAg and anti-HBe led to their concurrent detection when sera were tested by sensitive assays.更多还原